Clinical, Immunological, and Molecular Variability of RAG Deficiency: A Retrospective Analysis of 22 RAG Patients

Purpose We described clinical, immunological, and molecular characterization within a cohort of 22 RAG patients focused on the possible correlation between clinical and genetic data. Methods Immunological and genetic features were investigated by multiparametric flow cytometry and by Sanger or next generation sequencing (NGS) as appropriate. Results Patients represented a broad spectrum of RAG deficiencies: SCID, OS, LS/AS, and CID. Three novel mutations in RAG1 gene and one in RAG2 were reported. The primary symptom at presentation was infections (81.8%). Infections and autoimmunity occurred together in the majority of cases (63.6%). Fifteen out of 22 (68.2%) patients presented autoimmune or inflammatory manifestations. Five patients experienced severe autoimmune cytopenia refractory to different lines of therapy. Total lymphocytes count was reduced or almost lacking in SCID group and higher in OS patients. B lymphocytes were variably detected in LS/AS and CID groups. Eighteen patients underwent HSCT permitting definitive control of autoimmune/hyperinflammatory manifestations in twelve of them (80%). Conclusion We reinforce the notion that different clinical phenotype can be found in patients with identical mutations even within the same family. Infections may influence genotype–phenotype correlation and function as trigger for immune dysregulation or autoimmune manifestations. Severe and early autoimmune refractory cytopenia is frequent and could be the first symptom of onset. Prompt recognition of RAG deficiency in patients with early onset of autoimmune/hyperinflammatory manifestations could contribute to the choice of a timely and specific treatment preventing the onset of other complications.

Emerging therapy options may help patients with RAG deficiency, especially those with severe immune dysregulation

New understandings about RAG1 and RAG2 gene expression have recently been gained. Rag expression molecular pathways are now known to support research on genetic variants that influence regulatory domains for lymphoid development. The RAG1/RAG2 complex might serve as a model for RAG alterations in vivo and in vitro recombination experiments. These data indicate the approximate relationship between RAG deficiency genotype and phenotype. Although this concept has not yet been proven, it is theorized that hypomorphic RAG mutations restrict RAG complexes and interfere with appropriate T-and B-cell development.A unique cellular and animal model has uncovered significant immunological and clinical insights regarding RAG mutations. Rag mutations causing below-normal levels of T and B cell development have considerable ramifications on thymic lymphostromal cross talk as well as on receptor editing in the bone marrow and on the survival of self-reactive peripheral B cells. These difficulties account for the higher occurrence of autoimmune symptoms in people and animals with hypomorphic RAG mutations. A study looking at human T cell development used the in vitro methodology to investigate RAG-mutant mice. Finally, emerging therapy options may help patients with RAG deficiency, especially those with severe immune dysregulation. Useful therapy could benefit other people with immune inborn errors as well.New exploratory approaches are emerging to help cure RAG deficiency. These techniques have offered a distinct understanding of RAG deficiencies employing artificial thymic organoids (ATOs) and induced pluripotent stem cells, respectively (iPSCs). novel possible therapeutic approaches, such as lentiviral vector‐mediated gene therapy, gene editing, and nongenotoxic conditioning regimens for hematopoietic stem cell transplantation (HSCT).

Clinical, Immunological and Molecular Variability of RAG Deficiency: a Retrospective Analysis of 22 RAG Patients

Purpose RAG deficiency is associated with a variety of clinical phenotypes. We described clinical, immunological and molecular characterization within a cohort of 22 RAG patients focused on the possible correlation between clinical and genetic data. Methods Immunological and genetic features were investigated by Multiparametric Flow Cytometry and by Sanger or Next generation sequencing (NGS) respectively. Results Patients represented a broad spectrum of RAG deficiencies: SCID n=8, OS n=6, LS/AS n=4 and CID n=4. Four novel mutation in RAG1 gene and one in RAG2 were reported. The primary symptom at presentation were infections (81.8%). Infections and autoimmunity occurred together in the majority of cases (63.6%). Fifteen out of 22 (68.2%) patients presented autoimmune/hyperinflammatory manifestations. Four patients experienced severe autoimmune cytopenia refractory to different lines of therapy. Total lymphocytes count was reduced or almost lacking in SCID group. CD4 cells count was higher in OS patients. B lymphocytes were variably detected in AS and CID groups. Eighteen patients underwent HSCT permitting definitive control of autoimmune/hyperinflammatory manifestations in twelve of them (80%). Conclusion RAG deficiency still represents a challenge in the tracing of effective management and follow-up, notably considering the inability to predict the disease course in atypical cases. Immune dysregulation manifestations are common features often refractory to conventional medical management. Severe and early autoimmune refractory cytopenia is frequent and could be the first symptom of onset. Prompt recognition of RAG deficiency in patients with early onset of autoimmune/hyperinflammatory manifestations could contribute to the choice of a timely and specific treatment preventing the onset of other complications.

An AIREless Breath: Pneumonitis Caused by Impaired Central Immune Tolerance

Autoimmune-polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), a monogenic disorder caused by biallelic mutations in the AIRE gene, has historically been defined by the development of chronic mucocutaneous candidiasis together with autoimmune endocrinopathies, primarily hypoparathyroidism and adrenal insufficiency. Recent work has drawn attention to the development of life-threatening non-endocrine manifestations such as autoimmune pneumonitis, which has previously been poorly recognized and under-reported. In this review, we present the clinical, radiographic, autoantibody, and pulmonary function abnormalities associated with APECED pneumonitis, we highlight the cellular and molecular basis of the autoimmune attack in the AIRE-deficient lung, and we provide a diagnostic and a therapeutic roadmap for patients with APECED pneumonitis. Beyond APECED, we discuss the relevance and potential broader applicability of these findings to other interstitial lung diseases seen in secondary AIRE deficiency states such as thymoma and RAG deficiency or in common polygenic autoimmune disorders such as idiopathic Sjögren’s syndrome.

Innovative Cell-Based Therapies and Conditioning to Cure RAG Deficiency

Genetic defects in recombination activating genes (RAG) 1 and 2 cause a broad spectrum of severe immune defects ranging from early severe and repeated infections to inflammation and autoimmune manifestations. A correlation between in vitro recombination activity and immune phenotype has been described. Hematopoietic cell transplantation is the treatment of care; however, the availability of next generation sequencing and whole genome sequencing has allowed the identification of novel genetic RAG variants in immunodeficient patients at various ages, raising therapeutic questions. This review addresses the recent advances of novel therapeutic approaches for RAG deficiency. As conventional myeloablative conditioning regimens are associated with acute toxicities and transplanted-related mortality, innovative minimal conditioning regimens based on the use of monoclonal antibodies are now emerging and show promising results. To overcome shortage of compatible donors, gene therapy has been developed in various RAG preclinical models. Overall, the transplantation of autologous gene corrected hematopoietic precursors and the use of non-genotoxic conditioning will open a new era, offering a cure to an increasing number of RAG patients regardless of donor availability and severity of clinical conditions.

Immune dysregulation in patients with RAG deficiency and other forms of combined immune deficiency

Traditionally, primary immune deficiencies have been defined based on increased susceptibility to recurrent and/or severe infections. However, immune dysregulation, manifesting with autoimmunity or hyperinflammatory disease, has emerged as a common feature. This is especially true in patients affected by combined immune deficiency (CID), a group of disorders caused by genetic defects that impair, but do not completely abolish, T-cell function. Hypomorphic mutations in the recombination activating genes RAG1 and RAG2 represent the prototype of the broad spectrum of clinical and immunological phenotypes associated with CID. The study of patients with RAG deficiency and with other forms of CID has revealed distinct abnormalities in central and peripheral T- and B-cell tolerance as the key mechanisms involved in immune dysregulation. Understanding the pathophysiology of autoimmunity and hyperinflammation in these disorders may also permit more targeted therapeutic interventions.